The long-term goal of this research is to understand the molecular development of the nuclei and circuitry of the brainstem oculomotor control system. The focus of this project is on the regulatory mechanisms underlying oculomotor neuron production in the midbrain oculomotor complex (OMC) and the hindbrain trochlear nucleus (TrN). The proposed experiments will be carried out in chick embryos, which have the experimental advantage of site-directed transgenesis by electroporation and which share with other birds a highly organized OMC composed of discrete subnuclei with distinct extraocular muscle targets. The first set of experiments assesses the roles of homeodomain transcription factors in specifying oculomotor neuron identity and generating discrete OMC subnuclei. These experiments include a molecular dissection of the function of the CFEOM2 candidate gene PHOX2A. The second set of experiments investigates the molecular mechanisms that regulate the specification of midbrain progenitor cells to an oculomotor neuron fate, with a focus on the roles of NKX6 and basic helix-loop-helix transcription factors in these processes. The third set of experiments is based on our findings of pronounced transcription factor heterogeneity within the TrN and OMC subnuclei. We will investigate the anatomical correlate of this heterogeneity, focusing on the specific hypothesis that these molecular divisions identify motor neurons with distinct targets in the oculomotor plant. Study of the mechanisms that govern OMC and TrN development may provide insight into genetic diseases of the oculomotor system, most notably those such as CFEOM in which specific pools of oculomotor neurons are lost. More generally, understanding of the unique molecular specification of oculomotor cell types may suggest novel therapeutic strategies, including stem cell-based approaches, for the treatment of oculomotor disorders, and may give insight to the relative resistance of the oculomotor unit to some motor neuron diseases.